Intact epithelial barrier (e.g., skin) is important for health, functioning to exclude exogenous chemicals, antigens, and pathogens from the body (De Benedetto et al., “Skin Barrier Disruption: A Requirement for Allergen Sensitization?,” J. Invest. Dermatol. 132:949-963 (2012); O'Neill et al., “Tight Junction Proteins and the Epidermis,” Exp. Dermatol. 20:88-91 (2011); Kubo et al., “Epidermal Barrier Dysfunction and Cutaneous Sensitization in Atopic Diseases,” J. Clin. Invest. 122:440-447 (2012)). As such, it impedes transdermal delivery of therapeutic agents and vaccines. To address this issue, there has been a sustained and extensive effort to develop cell-penetrating peptides to facilitate transdermal delivery (Milletti, “Cell-Penetrating Peptides: Classes, Origin, and Current Landscape,” Drug Discov. Today 17:850-860 (2012); Stanzl et al., “Fifteen Years of Cell-Penetrating, Guanidinium-Rich Molecular Transporters: Basic Science, Research Tools, and Clinical Applications,” Acc. Chem. Res. 46(12):2944-54 (2013)). While this effort has produced many notable successes, an alternative strategy is to reversibly weaken the interactions between cells, thereby enabling paracellular delivery without the use of cell penetrating peptides.
The paracellular barrier in epithelial cell layers is sealed via tight junctions (“TJs”). In the skin, TJs form in the stratum granulosum (“SG”) (Yoshida et al., “Functional Tight Junction Barrier Localizes in the Second Layer of the Stratum Granulosum of Human Epidermis,” J. Dermatol. Sci. 71(2):89-99 (2013)) and may even persist into the stratum corneum (“SC”) (Haftek et al., “Compartmentalization of the Human Stratum Corneum by Persistent Tight Junction-Like Structures,” Exp. Dermatol. 20:617-621 (2011); Sugawara et al., “Tight Junction Dysfunction in the Stratum Granulosum Leads to Aberrant Stratum Corneum Barrier Function in Claudin-1-Deficient Mice,” J. Dermatol. Sci. 70(1):12-18 (2013)). TJs and the SC act in concert to form the epidermal barrier.
TJs form strands of protein assemblies in an equatorial belt around epithelial cells. Claudins bridge the actin cytoskeleton on the cytoplasmic face (through zona occludens proteins in the TJ plaque), span the plasma membrane, and interact with claudins on neighboring cells. At least 23 mammalian claudins have been identified thus far (Van Itallie et al., “Claudin Interactions In and Out of the Tight Junction,” Tissue Barriers 1(3): e25247 (2013)). Claudins are tetraspannins, with two extracellular loops and a long cytoplasmic C-terminal domain. At the cell surface, claudins form dimers through their extracellular domains that assemble with those on the opposing cell membrane, forming strands embedded in both cell membranes (Kaufmann et al., “Visualization and Quantitative Analysis of Reconstituted Tight Junctions Using Localization Microscopy,” PLoS ONE 7:e31128 (2012)). The number and character of these strands determines the strength of the paracellular barrier. Differential expression of claudin family members tailors the permeability characteristics for each tissue barrier (gut v. skin v. blood-brain barrier) (Anderson et al., “Physiology and Function of the Tight Junction,” Cold Spring Harb Perspect Biol. 1(2):a002584-a002584 (2009)). Several other membrane proteins including occludin (“Ocln”), tricellulin, and JAM-A also participate in the TJ complex and contribute to barrier function and regulation (Kirschner et al., “Contribution of Tight Junction Proteins to Ion, Macromolecule, and Water Barrier in Keratinocytes,” J. Invest. Dermatol. 133:1161-1169 (2013); Cording et al., “In Tight Junctions, Claudins Regulate the Interactions Between Occludin, Tricellulin and Marveld3, Which, Inversely, Modulate Claudin Oligomerization,” J. Cell Sci. 126:554-564 (2013)).
Claudin 1 (“Cldn1”) is highly expressed in lung epithelium, epidermal keratinocytes, and dendritic cells that populate the epidermis (Kast et al., “The Broad Spectrum of Interepithelial Junctions in Skin and Lung,” J. Allergy Clin. Immunol. 130:544-546 (2012); Kubo et al., “External Antigen Uptake by Langerhans Cells With Reorganization of Epidermal Tight Junction Barriers,” J. Exper. Med. 206:2937-2946 (2009)). Increasing Cldn1 expression in vitro enhances the paracellular permeability barrier (Pfeiffer et al., “Claudin-1 Induced Sealing of Blood—Brain Barrier Tight Junctions Ameliorates Chronic Experimental Autoimmune Encephalomyelitis,” Acta Neuropathol. 122:601-614 (2011); Inai et al., “Claudin-1 Contributes to the Epithelial Barrier Function in MDCK Cells,” European J. Cell Biol. 78:1-7 (1999)). In humans, a heritable Cldn1 deficiency has been reported in neonatal ichthyosis-hypotrichosis-sclerosing cholangitis syndrome, which results in severe ichthyosis and neonatal cholestasis (Hadj-Rabia et al., “Claudin-1 Gene Mutations in Neonatal Sclerosing Cholangitis Associated With Ichthyosis: A Tight Junction Disease,” Gastroenterology 127:1386-1390 (2004)). Mice lacking Cldn1 die of dehydration within one day of birth, indicating that the protein is essential for skin barrier (Yoshida et al., “Functional Tight Junction Barrier Localizes in the Second Layer of the Stratum Granulosum of Human Epidermis,” J. Dermatol. Sci. 71(2):89-99 (2013); Furuse et al., “Claudin-Based Tight Junctions are Crucial for the Mammalian Epidermal Barrier: A Lesson From Claudin-1-Deficient Mice,” J. Cell Biol. 156:1099-1111 (2002)). Similarly, patients with atopic dermatitis (“AD”) have greater transepidermal water loss and paracellular permeability in their nonlesional skin, and also have markedly reduced Cldn1 expression (De Benedetti, “Tight Junction Defects in Patients With Atopic Dermatitis,” J. Allergy Clint. Immune. 127:773-786.e7 (2010)).
Synthetic peptides derived from the sequence of the extracellular loops of TJ proteins (claudins and Ocln) have been shown to disrupt barrier function at high concentration by several research groups (Wong et al., “A Synthetic Peptide Corresponding to the Extracellular Domain of Occludin Perturbs the Tight Junction Permeability Barrier,” J. Cell Biol. 136:399-409 (1997); Mrsny et al., “A Key Claudin Extracellular Loop Domain Is Critical for Epithelial Barrier Integrity,” Am. J. Path. 172:905-915 (2008); Baumgartner et al., “A d-Peptide Analog of the Second Extracellular Loop of Claudin-3 and -4 Leads to Mislocalized Claudin and Cellular Apoptosis in Mammary Epithelial Cells,” Chem. Biol. & Drug Des. 77:124-136 (2011); Zwanziger et al., “A Peptidomimetic Tight Junction Modulator to Improve Regional Analgesia,” Mol. Pharm. 9:1785-1794 (2012)). A peptide from the second extracellular loop of claudins 3 and 4 induces mislocalization of occludin and apoptosis (Baumgartner et al., “A d-Peptide Analog of the Second Extracellular Loop of Claudin-3 and -4 Leads to Mislocalized Claudin and Cellular Apoptosis in Mammary Epithelial Cells,” Chem. Biol. & Drug Des. 77:124-136 (2011); Beeman et al., “Occluding Is Required for Apoptosis When Claudin—Claudin Interactions are Disrupted,” Cell Death Dis. 3:e273 (2012)). In T84 (transplantable human carcinoma) cells, application of 25 μM of the rat Cldn1 (53-80) peptide was shown to inhibit calcium-induced TJ formation, and 200 μM was able to disrupt intact TJs (Mrsny et al., “A Key Claudin Extracellular Loop Domain Is Critical for Epithelial Barrier Integrity,” Am. J. Path. 172:905-915 (2008)). Cysteines were not required for TJ disruption. Rat Cldn1 (53-81, C54,64S) peptide disrupted Caco-2, HEK-293, and rat perineural TJ barriers in vivo at 200-300 μM (Zwanziger et al., “A Peptidomimetic Tight Junction Modulator to Improve Regional Analgesia,” Mol. Pharm. 9:1785-1794 (2012)). Zwanziger, et al. noted that this peptide has β-sheet structure when solubilized with SDS as evidenced by circular dichroism. Rat Cldn1 (53-81, C54,64S) peptide co-localizes with Cldn1 and is internalized by subconfluent HEK-293 cells (Zwanziger et al., “Claudin-Derived Peptides are Internalized Via Specific Endocytosis Pathways,” Ann. NY Acad. Sci. 1257:29-37 (2012)).
A relatively high concentration of tested peptides was required to induce alteration in TJ appearance and increase paracellular permeability in these prior studies. No claudin-derived peptides capable of disrupting TJs at low or sub micromolar concentrations have been identified previously. Other than knowledge of the claudin-1 second loop domain and its amino acid sequence, structural features responsible for disruption have not been identified to date.
Further, transepithelial (including transdermal) delivery of large and/or hydrophilic therapeutics or antigens is not straightforward due to, e.g., the dual barrier functions of skin and mucosa. A small subset of small molecule drugs can readily cross epithelial cell barriers via transcellular transport by virtue of their small size and hydrophobicity. Larger or more hydrophilic molecules do not spontaneously cross cellular membranes and are excluded by paracellular tight junction barriers. Reversible, controlled barrier disruption that would enable noninvasive delivery of drugs and needle-free vaccines is particularly desirable for peptide/protein based therapeutics or antigens that do not readily cross cellular membranes.
Disclosed herein are peptides and formulations directed to overcoming these and other limitations in the art.